Certain nucleic acid probes comprising a single-stranded oligonucleotide and a pair of fluorescent dyes to induce FRET including a donor dye fluorescein and an acceptor dye X-rhodamine are known in the art [Jikken Igaku (Experimental Medicine), 15, e7, 728-733 (1997)]. These nucleic acid probes are used in monitoring of polymerase chain reaction (PCR) and are not intended to direct determination of nucleic acids.
Some nucleic acid probes have been used for detecting a target nucleic acid (Biochemistry, 34, 285-292 (1995). In this case, the nucleic acid probe emits fluorescence at a low intensity, as a result of FRET, before hybridization with the target nucleic acid. Specifically, the fluorescein is prevented from emitting fluorescence and can emit fluorescence only at a low intensity. In contrast, the X-rhodamine is not so prevented from emitting fluorescence but emits fluorescence at an intensity lower than that of the prevented fluorescence emission of the fluorescein. Upon hybridization with the target nucleic acid, the probe changes in its conformation such that FRET disappears. As a result, the fluorescein emits fluorescence at an increasing intensity to thereby increase the fluorescence intensity of the whole reaction system. However, if a nucleic acid probe does not change in its conformation after hybridization, the fluorescence intensity does not change, and such a nucleic acid probe is not suitable for use in nucleic acid determination.
The present inventors have developed a nucleic acid probe, namely, “a fluorescence quenching nucleic acid probe” that decreases its fluorescence emission after hybridization with a target nucleic acid without the aid of another nucleic acid probe (Nucleic acid, 29, No. 6 e34 (2001); EP 1 046 717 A9; Japanese Patent Publication No. 2001-286300A). The nucleic acid probe comprises a single-stranded oligonucleotide labeled with a fluorescent dye in an end region. The fluorescent dye and the base sequence of the oligonucleotide are designed such that the fluorescence intensity decreases upon hybridization with the target nucleic acid. Use of the nucleic acid probe makes it possible to determine precisely, easily and in short time the target nucleic acid in a trace amount. However, this conventional method utilizes fluorescence quenching due to interaction between base-pair complex of G (guanine) and C (cytosine) combined through a hydrogen bond and the fluorescent dye upon the formation of a double-stranded oligonucleotide. In other words, the method utilizes fluorescence quenching due to emission energy transfer from the fluorescent dye to the complex. However, relatively a few type of fluorescent dye is subjected to quenching action according to this mechanism, and the method is limited in fluorescent colors that are usable.
When an assay system contains plural types of target nucleic acids, fluorescence colors of the number of color type(s) equal to that of the types of the target nucleic acids must be used to determine the target nucleic acids in parallel. In other words, nucleic acid probes emitting fluorescence of different colors in the number of type(s) equal to that of the types of the target nucleic acids are needed. In enlargement of the number of fluorescent color type(s) in the conventional method, the number of type(s) of fluorescent dyes are needed to be larger, which fluorescent dyes are capable of inducing interaction with the G-C hydrogen bonding pair and are different in emitting fluorescence colors. However, the types of fluorescent colors applicable to the conventional method are limited and thereby the method is limited in determining plural (specifically three or more) types of target nucleic acids in parallel. In addition, that types of fluorescent dyes are different in the conventional method means that exciting wavelengths different in types also are needed, thus requiring different types of excitation light sources to excite all the fluorescent dyes. The conventional method therefore requires an apparatus of a large-scale and is not economical.
Under these circumstances, it is an object of the present invention to provide a novel nucleic acid probe for nucleic acid determination (“novel nucleic acid probe for nucleic acid determination” may be hereinafter briefly referred to as “nucleic acid probe”), which includes a single-stranded oligonucleotide labeled with a pair of fluorescent dyes capable of inducing FRET and including a donor dye and an acceptor dye and can precisely and easily determine one or more types (i.e., one or plural types) of target nucleic acids in small amounts in parallel in a short time using a simple analyzer (instrument). Another object of the present invention is to provide a novel method for nucleic acid determination using the nucleic acid probe (hereinafter briefly referred to as “nucleic acid determination method”), a reagent kit for use in the method, a method for determining polymorphism and/or mutation of target nucleic acids, and an assay kit for use in the method just mentioned above.